This proposal continues our research in the development of high performance liquid chromatography (HPLC) to problems of biochemical importance. There are two main objectives. First, the use of liquid chromatography/mass spectrometry for the structural analysis of peptides will be developed. The work is to be undertaken in collaboration with Dr. Paul Vouros, a mass spectroscopy specialist. In this proposal we shall develop specific hydrolytic procedures for fragmentation of oligopeptides and proteins and HPLC separation procedures. In particular, a novel peptide retention index is proposed for retention characterization, based on specific indices of amino acid residues. A variety of derivatized peptides will be examined, especially permethylated N-acetyl methyl esters. This procedure will be used for peptide sequencing and the analysis of hemoglobin variants and modified proteins. The second project continues our effort to develop new packings for the HPLC separation of proteins using wide pore diameter (500 Angstroms) silica as support matrix. Here we shall focus on reversed phase liquid chromatography and study in detail the adsorption of proteins on bonded n-alkyl phases. This work will be performed using full coverage bonded phases as well as low density phases in which the n-alkyl group is diluted by a hydrophilic bonded chain. Other studies will involve the synthesis of polar and ionic bonded phases, particularly for ion exchange chromatography of proteins. Again, diluted phases will be examined in depth. Finally, novel bonded phases employing metal chelates will be synthesized and studied for the separation of proteins based on ligand exchange.